This project is directed to develop technology to identify antigens recognized by pathogen-specific class-l MHC restricted T cells by systematic screening of all pathogen protein coding genes, its ORFeome. The system is based on the observation that phagolysosomal destruction within antigen presenting cells (APCs) of E. co// expressing cytoplasmic listeriolysin (cytoLLO) results in leakage of phagosomal contents into the ARC cytosol and access of antigens to the host cell cytosolic MHC class I antigen processing pathway resulting in presentation of the antigen in the context of MHC class I molecules. We propose to focus on immunity to S. Typhi, a pathogen of great public health importance. Live, attenuated vaccine strains of Salmonella have the potential to be a cost-effective prophylactic measure in combating this disease, yet the antigens important in protection are largely unknown. Thus, the identification of putative S. Typhi proteins that might be involved in protection, by screening of the S. Typhi ORFeome, will allow us to explore our hypothesis that the increased efficacy of 4 doses of the oral Ty21 a typhoid vaccine as compared to a single dose is in part due to the repertoire of S. Typhi specific CD8+ T cells in vaccinees. One of the novel features of the proposed technology is an ability to confirm that S. Typhi proteins are expressed and able to be presented by APCs in the context of class-l molecules by fusing them with positive epitope controls. In addition, rather than co-expressing cytoLLO and a candidate antigen in the same co// cell, we propose to co-infect ARC with two different strains of E. co//, one expressing cytoLLO and the other the antigen of interest as this system allows greater flexibility in use of the ORFeome for other purposes. This proposal brings together collaborators with expertise in cellular immunology, genomics and high throughput assays systems with the express purpose of tackling an important global health problem via improved vaccine development.